Proline derivatives

ABSTRACT

The present invention relates to particular forms of a novel 1-substituted-N- 2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide which are inhibitors of human leukocyte elastase (HLE), also known as human neutrophil elastase (HNE), making them useful whenever such inhibition is desired, such as for research tools in pharmacological, diagnostic and related studies and in the treatment of diseases in mammals in which HLE is implicated. The invention also includes pharmaceutical compositions containing such forms, processes for preparing the forms and intermediates useful in the synthesis of the forms.

This is a divisional application of application Ser. No. 08/595,692filed Feb. 2, 1996 now U.S. Pat. No. 5,686,628.

The present invention relates to novel proline derivatives, and moreparticularly to particular forms of a novel 1-substituted-N-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide derivativewhich are inhibitors of human leukocyte elastase (HLE), also known ashuman neutrophil elastase (HNE), which are of value, for example, as aresearch tool in pharmacological, diagnostic and related studies and inthe treatment of diseases in mammals in which HLE is implicated. Forexample, HLE has been implicated causally in the pathogenesis of acuterespiratory distress syndrome (ARDS), rheumatoid arthritis,atherosclerosis, pulmonary emphysema, and other inflammatory disorders,including airway inflammatory diseases characterized by increased andabnormal airway secretion such as acute and chronic bronchitis andcystic fibrosis. Also, HLE has been causally implicated in certainvascular diseases and related conditions (and their therapy) in whichneutrophil participation is involved or implicated, for example, inhemorrhage associated with acute non-lymphocytic leukemia, as well as inreperfusion injury associated with, for example, myocardial ischaemiaand related conditions associated with coronary artery disease such asangina and infarction, cerebrovascular ischaemia such as transientischaemic attack and stroke, peripheral occlusive vascular disease suchas intermittent claudication and critical limb ischaemia, venousinsufficiency such as venous hypertension, varicose veins and venousulceration, as well as impaired reperfusion states such as thoseassociated with reconstructive vascular surgery, thrombolysis andangioplasty. The invention also concerns methods of treating one or moreof these disease conditions and the use of one or more of the particularforms of the novel dervative in the manufacture of a medicament for usein one or more of said conditions. The invention further concernspharmaceutical compositions containing one or more of the particularforms of the novel derivative as active ingredient, as well as processesfor the manufacture of the particular forms of the novel derivative,novel intermediates useful in said processes and methods for thepreparation of said intermediates.

Because of HLE's apparent role, there has been considerable researcheffort in recent years towards the development of HLE inhibitors. InU.S. Pat. No. 4,910,190 is disclosed a series of structurally relatedpeptidoyl trifluoromethane derivatives which are HLE inhibitors. We havenow discovered that particular forms of the novel 1-substituted-N-2-methyl-1-(trifluoroacetyl)-propyl!-pyrrolidine-2-carboxamidederivative of formula I (set out hereinafter) are unexpectedly potentinhibitors of HLE.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the X-ray powder diffraction spectrum of a typical sampleof the crystalline form of the SSS diastereoisomer of formula I, whensubstantially or essentially pure and in hydrated form, referred to asForm A.

FIG. 2 shows the X-ray powder diffraction spectrum of FIG. 1 on anexpanded scale.

FIG. 3 shows the X-ray powder diffraction spectrum of a typical sampleof the crystalline form of the SSS diastereoisomer of formula I, whensubstantially or essentially pure and in hydrated form, referred to asForm B.

FIG. 4 shows the X-ray powder diffraction spectrum of a typical sampleof the SSS diastereoisomer of formula I in the "ketone" form.

FIG. 5 shows the infra-red spectrum of a typical sample of thecrystalline form of the SSS diastereoisomer of formula I referred to asForm A.

FIG. 6 shows the infra-red spectrum of a typical sample of thecrystalline form of the SSS diastereoisomer of formula I referred to asForm A obtained using a 2% dispersion of the sample in potassiumbromide.

FIG. 7 shows the infra-red spectrum of a typical sample of thecrystalline form of the SSS diastereoisomer of formula I referred to asForm B obtained using conditions similar to the conditions used for FIG.6.

FIG. 8 shows the infra-red spectrum of a typical sample of the SSSdiastereoisomer of formula I in substantially the "ketone" form obtainedusing conditions similar to the conditions used for FIG. 6.

According to one aspect of the invention, there is provided the compound(S)-1- (S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide, or asolvate thereof, both in the form of a diastereomeric mixture of (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(S)-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide (or asolvate thereof) and (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(R)-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide (or asolvate thereof) and in the form of the substantially or essentiallypure diastereoisomer (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(S)-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide or asolvate thereof.

It will be appreciated that the compound of formula I has three chiralcentres (identified by * and # in formula I) and can therefore exist ineight different stereomeric forms, or as a diastereomeric mixture of twoor more of these forms. For example, the compound (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide is acompound of the formula I in which the two chiral centres identifiedby * have the S configuration and the third chiral centre identified by# has the RS configuration. The compound is therefore a diastereomericmixture comprising the diastereoisomer with the chiral centres marked *and # all having the S configuration, that is (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(S)-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide(hereinafter referred to as the "SSS diastereoisomer" of formula I andwhich may also be represented as shown in formula Ia in which athickened bond denotes a bond projecting from the plane of the paper)and the diastereoisomer with the chiral centres marked * having the Sconfiguration and that marked * having the R configuration, that is(S)-1- (S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(R)-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide(hereinafter referred to as the "SSR diastereoisomer" of formula I), orsolvates thereof. Such a diastereomeric mixture includes, for example, amixture containing approximately equal amounts of the SSS and SSRdiastereoisomers, i.e. the SSS:SSR ratio is about 1:1. For example,diastereomeric mixtures comprising SSS and SSR diastereoisomers inratios of 53:47 and 47:53 (SSS:SSR) have been obtained. Particular formsof the compound of formula I which are preferred are diastereomericmixtures which are enriched in the SSS diastereoisomer, i.e. the ratioof SSS:SSR is greater than 1:1. An especially preferred form of thecompound is substantially or essentially pure SSS diastereoisomer, thatis SSS diastereoisomer containing less than 5% (more particularly lessthan 3% and preferably less than 2%) of other diastereoisomers.

It will be appreciated that the SSS diastereoisomer of formula I mayalso form a diastereomeric mixture with one or more other forms offormula I, for example (S)-1- 2-methoxycarbonylamino-3-methylbutyryl!-N-(S)-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide (adiastereomeric mixture of the SSS and RSS forms of formula I) or 1-(S)-2-methoxycarbonylamino-3-methylbutyryl!-N-(S)-2-methyl-1-(trifluoroacetyl)propyl!pyrrolidine-2-carboxamide (adiastereomeric mixture of the SSS and SRS forms of formula I) may beobtained. These particular diastereomeric mixtures, and otherdiastereomeric mixtures containing about 50% or more of the SSSdiastereoisomer together with one or more other possiblediastereoisomers with different configurations at the chiral centresmarked by * and # in formula I are therefore further aspects of thepresent invention.

A diastereomeric mixture of the SSS and SSR diastereoisomers may existin an amorphous, non-crystalline form or in a crystalline form,dependent on the ratio of SSS:SSR diastereoisomers present. A preferreddiastereomeric mixture is one which can be isolated in crystalline form,which is particularly advantageous in the manufacture of the compound,or formulations thereof, to the purity levels and uniformity requiredfor regulatory approval. It will be appreciated that it is extremelydifficult to obtain a compound which is a single diastereoisomercompletely free of the other possible diastereomeric forms, particularlya compound which has three chiral centres. The present inventiontherefore includes a crystalline form of the SSS diastereoisomer offormula I, or solvate thereof, which contains other possiblediastereoisomers with different configurations at the chiral centresindicated by * and # in formula I. It has been found that a crystallinediastereomeric mixture of the SSS and SSR diastereoisomers or a hydratethereof, can be obtained which is substantially or essentially adiastereomeric mixture of SSS and SSR diastereoisomers in a ratio(SSS:SSR) of 65:35 or greater, i.e. it contains 35% or less of the SSRdiastereoisomer. The present invention therefore includes a crystallineform of the compound of formula I, or a solvate thereof, with a contentof at least 65% of the SSS diastereoisomer. Preferably the crystallinediastereomeric mixture has, for example, a ratio of SSS:SSR which is80:20 or greater, for example 95:5 or greater, and especially 98.5:1.5or greater. An especially preferred form of the compound of theinvention is crystalline SSS diastereoisomer which is substantially oressentially pure, i.e. it contains less than 5% of otherdiastereoisomers, for example less than 5% of the SSR diastereoisomer,preferably less than 3% of the SSR diastereoisomer, and more preferablyless than 2% of the SSR diastereoisomer.

An amorphous or crystalline diastereomeric mixture of SSS and SSR forms,or substantially or essentially pure SSS diastereoisomer, exist in aform which is substantially or essentially free of solvent (hereinafterreferred to as the "ketone" form and as illustrated in Formula Ia forthe pure SSS diastereoisomer), or as a solvated, for example, hydratedform, or as a mixture of the ketone and solvated (hydrated) forms. Thehydrated form may exist, for example, as a gem-diol of thetrifluoroketone functionality, that is as a compound of the formula Ib(set out hereinafter) for substantially or essentially pure SSSdiastereoisomer, or as a compound of the formula Ic (set outhereinafter), or as a form which incorporates a water molecule as partof the crystal lattice, or mixtures of such forms. The compounds offormula Ib or Ic may, for example, be further hydrated.

It will be appreciated that the degree of hydration of a diastereomericmixture or substantially or essentially pure SSS diastereoisomer may beexpressed as a ratio of hydrate to ketone forms. For example, anamorphous, non-crystalline diastereomeric mixture of SSS and SSR formshas been isolated in which the ratio of hydrated form to ketone formvaries, for example, from about 30:70 (i.e. enriched in the ketone form)to about 95:5 or greater (i.e. substantially or essentially in thehydrated form), including such ratios as about 50:50 and about 60:40.Crystalline forms have, for example, been obtained which have an SSS:SSRratio of about 95:5 together with a hydrate:ketone ratio of about 80:20and which have an SSS:SSR ratio of about 65:35 or greater (such as98.5:1.5) and are substantially or essentially in the hydrated form.Crystalline hydrates of substantially or essentially pure SSSdiastereoisomer containing approximately 4.1% (w/w) and 7.8% (w/w) ofwater have also been obtained. Such particular forms are further aspectsof the invention. It is to be further understood that the presentinvention also encompasses any ketal or hemiketal (or mixture thereof)of a diastereomeric mixture or of a form of the SSS diastereoisomer, orof a solvate thereof, referred to herein which is converted to thegem-diol in vivo, for example by hydrolysis or enzymatic cleavage (andwherein the residue is pharmaceutically acceptable). The presentinvention also includes any tautomer or pro-drug of the SSSdiastereoisomer or a solvate thereof.

It will be appreciated that the compound of formula Ib may be referredto as the gem-diol form of the compound of formula Ia or by the chemicalname (S)-1- (S)-2-(methoxycarbonylamino)-3-methylbutyryl)-N-(S)-2-methyl-1-(2,2,2-trifluoro-1,1-dihydroxyethyl)-propyl!pyrrolidine-2-carboxamide.It will also be appreciated that an alternative name for the compound offormula Ia is methyl N- (1S)-1-((2S)-2-N-((1S)-2-methyl1-(2,2,2-trifluoroacetyl)propyl)-carbamoyl!pyrrolidine-1-ylcarbonyl)-2-methylpropyl!carbamateand an alternative name for the compound of formula Ib is methyl N-((S)-1-((2S)-2-N-((S)-3,3,3-trifluoro-2,2-dihydroxy-1-isopropylpropyl)carbamoylpyrrolidin-1-yl-carbonyl)-2-methylpropyl!-carbamate.

The melting point of crystalline SSS diastereoisomer containing SSRdiastereoisomer generally depends on the level of SSR diastereoisomerpresent and the level of solvation (hydration). It may be determined byconventional procedures well known in the art, for example, bydifferential scanning calorimetry (DSC).

Preferably the crystalline SSS diastereoisomer is in a hydrated form.For example, hydrated forms of the SSS diastereoisomer have been foundwhich have an advantageous property that they are non-hygroscopic, forexample Form A and Form B referred to hereinafter. Thus a preferred formof the SSS diastereoisomer is a crystalline form containing less than 5%(preferably less than 3% and especially less than 2%) of the SSRdiastereoisomer and substantially or essentially in a hydrated form.Such crystalline hydrated forms, for example Form A and Form B, havebeen found to possess good bioavailability and good solubility inaqueous buffer, which are both advantageous properties.

A particularly preferred crystalline form of the SSS diastereoisomer offormula I, when it is substantially or essentially pure and in ahydrated form, has an X-ray powder diffraction pattern including twomajor specific peaks at about 2θ=10.8° and 11.4°. This form (hereinreferred to as Form A) contains approximately 4.1% water. The X-raypowder diffraction pattern also includes less relatively intensespecific peaks occurring at about 2θ=15.4°, 16.8°, 18.2°, 18.6°, 20.6°,21.6°, 21.9°, 22.8° and 25.0°. The X-ray powder diffraction spectrum(XDS) of a typical sample of this form is shown in FIGS. 1 and 2hereinafter, where FIG. 2 shows the less intense peaks on an expandedscale. Further physical data suggests that this crystalline form issubstantially or essentially in the form of the diol of formula Ib.

A further preferred crystalline form of the SSS diastereoisomer offormula I, when it is substantially or essentially pure and in ahydrated form, has an X-ray powder diffraction pattern including a majorspecific peak at about 2θ=7.2°. This form (herein referred to as Form B)contains approximately 7.8% w/w (for example 7.3-8.3% w/w) of water. TheX-ray powder diffraction pattern also includes less relatively intensespecific peaks occurring at about 2θ=7.4°, 9.0°, 10.8°, 11.3°, 14.5°,15.9°, 17.8°, 18.1°, 19.7° and 22.5°. The XDS of a typical sample ofthis form is shown in FIG. 3 hereinafter. Further physical data suggeststhat this crystalline form is substantially or essentially themonohydrate of the diol of formula Ib.

When it is substantially or essentially pure and substantially oressentially free of solvent (i.e. in the "ketone" form), the SSSdiastereoisomer has an X-ray powder diffraction pattern including amajor specific peak at about 2θ=12.1. This pattern also includes lessrelatively intense peaks occurring at about 2θ=6.0°, 16.8° and 17.7°.The XDS of a typical sample of the "ketone" form is shown in FIG. 4hereinafter.

The X-ray powder diffraction spectra were determined, for example, usingScintag XDS-2000 X-ray diffractometer, with an EC&G solid-state photondetector, GLP Series (germanium) operated by a Microvax computer andusing the Diffraction Management System software supplied by ScintagInc., Sunnydale, Calif., USA. The X-ray tube used was a Cu K-alpha witha wavelength of 1.5406 A at 45 KV and 40 mA. The receiving slits wereset at 2 and 4 mm and the diverging slits set at 0.5 and 0.2 mm withrespect to the path of the incident beam. The spectra were obtained inthe continuous scan mode with a chopper increment of 0.02. Each samplewas exposed at 1 degree 2-theta per minute (running time was 38 minutes)and collected from 2 to 40 degrees 2-theta, to produce a trace ofspacings against intensity for this range.

For diffraction analysis the samples were packed into round aluminiumalloy sample pans with a diameter of 25 mm and depth of 2 mm. The powdersample was placed in the pan so that an amount in excess of the panvolume was present and subsequently leveled to the pan rim with a glassmicroscope slide. Silicon type-NBS 640b was used as an externalstandard.

Alternatively a Siemens D5000 X-ray diffractometer was used, recordingthe diffractogram in θ--θ mode over the range 2 to 40 degrees 2-thetawith 4 seconds exposure per 0.02° 2θ increment.

An infra-red spectrum was obtained for a typical sample of Form A. Theinfra-red spectrum was obtained by the solvent cast technique well knownin the art, from acetonitrile castings of a sample onto a salt windowfor analysis by direct transmission. The infra-red spectrum wasdetermined over the wave number range 4000 to 400 cm⁻¹. The infra-redspectrum is shown in FIG. 5. The spectrum of FIG. 5 includes sharp peaksat about 2968, 1762, 1721, 1690, 1632, 1525, 1447, 1207 and 1154 cm⁻¹.

The infra-red spectrum was also obtained for a typical sample of Form Ausing a Nicolet 20SXC FTIR spectrometer. The spectrum was obtained usinga 2% dispersion of the sample in potassium bromide. The infra-redspectrum is shown in FIG. 6 hereinafter. The spectrum of FIG. 6 includessharp peaks at about 3402, 3321, 3252, 3060, 2967, 2878, 1699, 1674,1629, 1535, 1532, 1446, 1271, 1258, 1249, 1175, 1152, 1118, 1089, 1029,1013, 1004, 635, 593 and 567 cm⁻¹. Using similar conditions, aninfra-red spectrum was obtained for a typical sample of Form B. Theinfra-red spectrum is shown in FIG. 7 hereinafter. The spectrum of FIG.7 includes sharp peaks at about 3428, 3304, 2971, 2875, 1708, 1682,1637, 1556, 1518, 1470, 1449, 1428, 1316, 1310, 1277, 1265, 1236, 1196,1175, 1144, 1120, 1081, 1036, 1005, 928, 818, 790 and 727 cm⁻¹. Usingsimilar conditions, an infra-red spectrum was obtained for a typicalsample of the SSS diastereoisomer in substantially the "Ketone" form.The infra-red spectrum is shown in FIG. 8. The spectrum of FIG. 8includes sharp peaks at about 3415, 3300, 2967, 2876, 1764, 1723, 1711,1695, 1686, 1634, 1527, 1445, 1356, 1286, 1234, 1213, 1139, 1105, 1061,1020, 774, 732 and 671cm⁻¹.

It will be understood that the 2θ values of the X-ray powder diffractionpatterns and the wavelengths of the infra-red spectra may vary slightlyfrom one machine to another and so the values quoted are not to beconstrued as absolute. For example, the two major specific peaks whichoccurred at about 2θ=10.8° and 11.4° for a typical sample of Form A whena Scintag XDS-2000 X-ray diffractometer was used, occurred at about2θ=10.6° and 11.2° respectively when a Siemens D5000 X-raydiffractometer was used (with the less intense peaks also occurring at aproportionately lower relative 2θ value).

It will be appreciated that the hydrogen atoms of the hydroxyl groups ofthe forms of formula Ib or Ic (or a hydrate thereof) are acidic and thatsuch compounds may therefore form crystallinepharmaceutically-acceptable salts, using conventional procedures, forexample with bases affording physiologically-acceptable cations, forexample alkali metal (such as sodium or potassium), alkali earth metalor organic amine salts. The invention therefore includes crystallinepharmaceutically-acceptable salts of the forms of formula Ib and Ic or ahydrate thereof.

The various forms of the compound of formula I referred to above, orsolvates (hydrates) thereof, may be obtained, for example, by thefollowing processes, which are further separate aspects of theinvention.

A non-crystalline (amorphous) diastereomeric mixture of SSS and SSRdiastereoisomers may be obtained by oxidation of the compound of formulaII with a suitable oxidising agent.

A suitable oxidising agent is one known in the art for the conversion ofa hydroxy group into a ketone group. Suitable oxidising agents andconditions include, for example, the use of oxalyl chloride, dimethylsulfoxide, and a tertiary amine; the use of acetic anhydride anddimethyl sulfoxide; the use of chromium trioxide pyridine complex indichloromethane; the use of hypervalent iodine reagent, such as1,1,1-triacetoxy-2,1-benzoxidol-3(3H)-one with trifluoroacetic acid indichloromethane; the use of excess dimethylsulphoxide and a watersoluble carbodiimide in the presence of dichloroacetic acid; or analkaline aqueous alkali metal permanganate, such as alkaline aqueouspotassium permanganate or sodium permanganate solution. Particularlysuitable oxidising agents are the latter two named, especially alkalineaqueous potassium or sodium permanganate solution, for example a mixtureof sodium hydroxide and potassium or sodium permanganate.

The compound of formula II may be obtained, for example, as shown inSchemes 1 and 2, using conventional procedures, or as illustrated in theExamples. Steps (a) to (d) of Scheme 1 may be carried out as describedin U.S. Pat. No. 5,194,588 or European Patent 189305. Step (e) iscarried out using conventional procedures for the formation of acarbamate from a primary amine, for example using a methylhalogenoformate, such as methyl chloroformate, in the presence of asuitable base such as triethylamine or N-methylmorpholine, and in asuitable solvent or diluent, for example a chlorinated hydrocarbon (suchas dichloromethane or chloroform) or an ethereal solvent (such astetrahydrofuran or dioxan), and at a temperature in the range of, forexample, -10° C. to 50° C., such as 0° C. to 30° C. The reaction stepsof Scheme 2 include conventional steps of protection (step (10)),deprotection or selective deprotection (steps (1), (3), (6), (8), (9)and (12)), coupling (steps (4), (5), (13) and (14) and carbamateformation (steps (2), (7) and (11)) well known in the art.

It will be appreciated that diastereomeric mixtures of SSS and RSSdiastereoisomers and of SSS and SRS diastereoisomers may be obtainedusing analogous procedures, with appropriate choice of L- or DL-valineor proline (or the protected derivatives thereof) as starting materialsand using (2R,3S)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanol at theappropriate coupling steps.

Substantially or essentially pure SSS diastereoisomer may be obtained,for example, by oxidation of (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(S)-2-methyl-1-((R)-2,2,2-trifluoro-1-hydroxyethyl)propyl!pyrrolidine-2-carboxamide(of formula IIa) with a suitable oxidising agent, such as one of theoxidising agents referred to above. The starting alcohol may be obtainedas shown in Scheme 2.

Crystalline forms of the SSS diastereoisomer containing 35% or less ofthe SSR diastereoisomer may be obtained from a non-crystalline(amorphous) diastereomeric mixture of the SSS and SSR diasteroisomers,containing the SSS and SSR diastereoisomers in approximately equalamounts (i.e. a ratio of about 1:1, typically 53:47 or 47:53) bycrystallisation from a suitable non-polar solvent, such as a mixture ofmethyl tert-butyl ether and hexane, preferably containing a smallquantity of water and optionally containing a small amount ofhydrochloric acid, for example 0-0.2 mole equivalents of 36% w/whydrochloric acid and 1-2.1 mole eqivalents of water. It is foundpreferable to add aqueous hydrochloric acid to the solvent ofcrystallisation when a non-crystalline diastereomeric mixture of SSS:SSRrato of 47:53 is used. To initiate crystallisation, seeding withcrystalline SSS diastereoisomer is preferred. The crystalline product isgenerally isolated as a mixture of hydrated and ketone form, typicallyin a ratio of about 80:20 (hydrated:ketone) or greater. A hydrated formor a mixture of ketone and hydrated forms may be converted to thesubstantially or essentially "ketone" form by drying in a vacuum oven(for example at about 50° C.). However, such a ketone form ishygroscopic.

Substantially or essentially pure crystalline forms of the SSSdiastereoisomer may be obtained by recrystallisation or repeatedrecrystallisation of crystalline forms of the SSS diasteroisomercontaining SSR diastereoisomer. Solvents or mixtures of solvents whichmay be used include, for example, butyl acetate, butyl acetate/hexane,acetone/water, acetone/hexane, acetone/petroleum fraction b.p. 100°-120°C., 1,2-dimethoxyethane/hexane, 1,2-dimethoxyethane/water/hexane, ethylacetate/water/hexane, ethyl acetate/hexane, water, dibutylether/hexane,dichloromethane/hexane, 1,2-dimethoxyethane/water, methanol/toluene,methyl tert-butyl ether/hexane, isopropanol/hexane andtetrahydrofuran/hexane. To obtain Form A, the first ten solvents ormixtures of solvents referred to above are preferred. Wet ethylacetate/hexane is particularly useful to obtain Form A. Particularlyuseful solvents or mixtures of solvents to obtain Form B are1,2-dimethoxyethane/water and water/methanol, although this form mayalso be obtained when ethyl acetate/water/hexane is used. Where hexaneis referred to herein, this includes isomers of hexane (such asiso-hexane) or mixtures thereof.

Substantially or essentially pure crystalline forms of the SSSdiastereoisomer may also be obtained by crystallisation of substantiallyor essentially pure SSS diastereoisomer isolated in a non-crystallineform (for example by oxidation of the compound of formula IIa), such asan oil, using similar solvents or mixtures of solvents referred toabove, especially a mixture of ethyl acetate, water and hexane.

Furthermore, Form A may also be obtained from Form B byrecrystallisation, for example as illustrated in Example 9. In additionthe crystalline "ketone" form (which is hygroscopic) may be obtainedfrom Form A, for example, as illustrated in Example 10.

The preparation of a ketal or hemi-ketal from a ketone is well known inthe art.

3-Amino-4-methyl-1,1,1-trifluoro-2-pentanol may be obtained as describedin U.S. Pat. No. 4,910,190 or as illustrated in the Examples.

A particularly advantageous procedure for the manufacture of(2R,3S)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanol, which is a furtheraspect of the present invention, comprises (as illustrated in Scheme 3):

(1) reaction of (2RS,3SR)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanol,or a salt thereof, with triphosgene or dimethyl carbonate in thepresence of a suitable base to give(4RS,5SR)-4-isopropyl-5-trifluoromethyloxazolidin-2-one; followed by

(2) reaction of (4RS,5SR)-4-isopropyl-5-trifluoromethyloxazolidin-2-one,or an alkali metal salt thereof, with (-)-menthyl chloroformate to give(4RS,5SR)-4-isopropyl-3-(1R,3R,4S)-3-p-menthyloxycarbonyl!-5-trifluoromethyloxazolidin-2-one andseparation of the (4S,5R)-4-isopropyl-3-(1R,3R,4S)-3-p-menthyloxycarbonyl!-5-trifluoromethyloxazolidin-2-oneisomer; followed by

(3) hydrolysis of (4S,5R)-4-isopropyl-3-1R,3R,4S)-3-p-menthyloxycarbonyl!-5-trifluoromethyloxazolidin-2-oneisomer under basic conditions, to give(2R,3S)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanol.

In Step (1), a suitable base is an aqueous alkali metal hydroxide, forexample sodium or potassium hydroxide. The reaction is generally carriedout in a suitable inert solvent or diluent, for example a hydrocarbonsuch as toluene. The reaction is exothermic and so the reaction isgenerally carried with external cooling maintaining the temperature atabout 0° C. to 50° C., for example at about ambient temperature.

In step (2), the reaction is carried out in a suitable solvent ordiluent, for example an ethereal solvent such as tetrahydrofuran.

Conveniently the oxazolidinone is converted to its alkali metal salt,for example using butyllithium at about -78° C., prior to addition ofthe (-)-menthyl chloroformate. On work-up, the desired (4S,5R)-isomercrystallises from the mixture of isomers and is collected by filtration.

In step (3), suitable conditions include, for example, the use of anaqueous solution of an alkali metal hydroxide (such as sodium orpotassium hydroxide) in an ethereal solvent or diluent, such as dioxan,at a temperature in the range of, for example, 60°-130° C. (such as90°-120° C.).

The utility of the compound of the invention may be demonstrated bystandard tests and clinical studies, including those described below.

Inhibition Measurements:

The potency of the compound of the invention (or a particular formthereof) to act as an inhibitor of human leukocyte elastase (HLE) on thelow molecular weight peptide substratemethoxy-succinyl-alanyl-alanyl-prolyl-valine-p-nitroanilide isdetermined as described in U.S. Pat. No. 4,910,190. The potency of thecompound is evaluated by obtaining a kinetic determination of thedissociation constant, K_(i), of the complex formed from the interactionof the inhibitor with HLE. The compound of Example 1 was found to have aK_(i) of 36 nM. The compound of Example 2 was found to have a K_(i) of 9nM.

Acute Lung Injury Model:

Animal models of emphysema include intratracheal (i.t.) administrationof an elastolytic protease to cause a slowly progressive, destructivelesion of the lung. These lesions are normally evaluated a few weeks toa few months after the initial insult. However, these proteases alsoinduce a lesion that is evident in the first few hours. The early lesionis first hemorrhagic, progresses to an inflammatory lesion by the end ofthe first 24 hours and resolves in the first week post insult. To takeadvantage of this early lesion, the following model may be used.

Hamsters are first lightly anesthetized with Brevital. Phosphatebuffered saline (PBS) pH 7.4, either alone or containing human leukocyteelastase (HLE), is then administered directly into the trachea.Twenty-four hours later the animals are killed and the lungs removed andcarefully trimmed of extraneous tissue. Following determination of wetlung weight, the lungs are lavaged with PBS and total lavagable red andwhite cells recovered are determined. The values for wet lung weights,total lavagable red cells and total lavagable white cells are elevatedin a dose-dependent manner following administration of HLE. Compoundsthat are effective elastase inhibitors can prevent or diminish theseverity of the enzyme-induced lesion resulting in lower wet lung weightand reduced values for total lavagable cells, both red and white,relative to administration of HLE alone. Compounds can be evaluated byadministering them intratracheally as solutions or suspensions in PBS,either with or at various times prior to the HLE challenge (400 μg), orby dosing them intravenously or orally as solutions at various timesprior to the HLE challenge (100 μg) to determine their utility inpreventing an HLE lesion. A solution of the compound of the invention(or a particular form thereof) may be conveniently prepared using 10%polyethylene glycol 400/PBS.

Acute Hemorrhagic Assay:

This assay relies on monitoring only the amount of hemorrhage in thelung following intratracheal administration of human neutrophil elastase(HNE). Hemorrhage is quantified by disrupting erythrocytes recovered inlung lavage fluid and comparing that to dilutions of whole hamsterblood. The screening protocol, similar to that described in Fletcher etal., American Review of Respiratory Disease (1990), 141, 672-677, is asfollows. Compounds demonstrated to be HNE inhibitors in vitro areconveniently prepared for dosing as described above for the Acute LungInjury Model. Male Syrian hamsters (fasted for 16-18 hours prior to use)are lightly anaesthetised with Brevital sodium (30 mg/kg i.p.). Thecompounds are then dosed intravenously or orally to the hamsters at afixed time, such as 30 or 90 min, prior to intratracheal administrationof 50 μg/animal of HNE in 300 μL phosphate buffered saline (PBS) pH 7.4.Four hours after enzyme administration, the animals are killed with anoverdose of pentobarbital sodium, the thorax opened and the lungs andheart removed and the lungs cleared of extraneous material. The excisedlungs are lavaged with three changes of 2 ml PBS via a tracheal cannula.The recovered lavages are pooled, the volumes (about 5 mL) are recordedand the lavages stored at 4° C. until assayed. For calculation of theamount of blood in each sample, the thawed lavages and a sample of wholehamster blood are sonicated to disrupt erythrocytes and appropriatelydiluted into individual wells of a 96-well microtiter plate. The opticaldensities (OD) of the disrupted lavages and blood samples are determinedat 540 nm. The (μL blood equivalents)/(mL lavage) are determined bycomparing the OD of the test samples with the OD of the standard curveprepared from whole hamster blood. The total μL equivalents of bloodrecovered is determined by multiplying recovered lavage volume by the(μL blood equivalents)/(mL lavage) for each sample. Results are reportedas % inhibition of HNE-induced hemorrhage with respect to PBS treatedcontrols when the test compound is given at a specified dose and timeprior to administration of HNE. The ED₅₀ for the compound of Example 1was found to be 4.5 mg/kg after oral dosing. The ED₅₀ for the compoundof Example 2 was found to be 1.9 mg/kg after oral dosing and 0.6 mg/kgafter i.v. administration.

No overt toxicity was observed when the compound of the invention wasadministered in the above in vivo tests.

It will be appreciated that the implications of a compound's activity inthe Acute Lung Injury Model or Acute Hemorrhagic Assay are not limitedto emphysema, but, rather, that the test provides evidence of general invivo inhibition of HLE.

According to a further feature of the invention, there is provided apharmaceutical composition comprising a pharmaceutically effectiveamount of the compound of the invention (or a particular form thereof),or a solvate thereof, and a pharmaceutically acceptable diluent orcarrier. As noted above, another feature of the invention is a method ofusing the compound of the invention (or a particular form thereof), or asolvate thereof, in the treatment of a disease or condition in a mammal,especially a human, in which HLE is implicated, such as those referredto hereinbefore, and particularly acute and chronic bronchitis,pulmonary emphysema, reperfusion injury, adult respiratory distresssyndrome, cystic fibrosis, or peripheral vascular disease (such ascritical limb ischaemia or intermittent claudication).

The compound of the present invention (or a particular form thereof) maybe administered to a warm-blooded animal, particularly a human, in needthereof for treatment of a disease in which HLE is implicated, in theform of a conventional pharmaceutical composition, for example asgenerally disclosed in U.S. Pat. No. 4,910,190. One mode ofadministration may be via a powdered or liquid aerosol. In a powderedaerosol, the compound of the invention (or a particular form thereof)may be administered in the same manner as cromolyn sodium via a`Spinhaler` (a trademark) turbo-inhaler device obtained from FisonsCorp. of Bedford, Mass. at a rate of about 0.1 to 50 mg per capsule, 1to 8 capsules being administered daily for an average human. Eachcapsule to be used in the turbo-inhaler contains the required amount ofthe compound of the invention (or the particular form thereof) with theremainder of the 20 mg capsule being a pharmaceutically acceptablecarrier such as lactose. In a liquid aerosol, the compound of theinvention (or a particular form thereof) may be administered using anebulizer such as, for example, a `Retec` (trademark) nebulizer, inwhich the solution is nebulized with compressed air. The aerosol may beadministered, for example, at the rate of one to about eight times perday as follows: A nebulizer is filled with a solution of the compound(or a particular form thereof), for example 3.5 mL of solutioncontaining 10 mg/mL; the solution in the nebulizer is nebulized withcompressed air; and the patient breathes normally (tidal volume) foreight minutes with the nebulizer in his mouth.

Alternatively, the mode of adminstration may be parenteral, includingsubcutaneous deposit by means of an osmotic pump or, preferably, oral.The compound of the invention (or a particular form thereof) may beconventionally formulated in an oral or parenteral dosage form bycompounding about 10 to 250 mg per unit of dosage with conventionalvehicle, excipient, binder, preservative, stabilizer, flavor or the likeas called for by accepted pharmaceutical practice, e.g. as described inU.S. Pat. No. 3,755,340. For parenteral administration, a 1 to 10 mLintravenous, intramuscular or subcutaneous injection would be givencontaining about 0.02 mg to 10 mg/kg of body weight of the compound ofthe invention (or a particular form thereof) 3 or 4 times daily. Theinjection would contain the compound of the invention (or a particularform thereof) in an aqueous isotonic sterile solution or suspensionoptionally with a preservative such as phenol or a solubilizing agentsuch as ethylenediaminetetraacetic acid (EDTA). For parenteraladministration or use in an aerosol, an aqueous formulation may beprepared, for example, by dissolving the compound (or a particular formthereof) in 5-10% polyethylene glycol 400/phosphate buffered saline,followed by aseptic filtration, and sterile storage using standardprocedures.

In general, the compound of the invention (or a particular form thereof)will be administered to humans at a daily dose in the range of, forexample, 5 to 100 mg of the compound (or a particular form thereof) byaerosol or 50 to 1000 mg intravenously or orally, or a combinationthereof. However, it readily will be understood that it may be necessaryto vary the dose of the compound (or a particular form thereof)adminstered in accordance with well known medical practice to takeaccount of the nature and severity of the disease under treatment,concurrent therapy, and the age, weight and sex of the patient receivingtreatment. It similarly will be understood that generally equivalentamounts of a solvated (for example, hydrated) form of the compound alsomay be used. Protocols for the administration of an HLE inhibitor andevaluation of the patients are described in the European PatentApplications with Publication Numbers 458535, 458536, 458537, and 463811for the treatment or prevention of cystic fibrosis, ARDS, bronchitis,and hemorrhage associated with acute non-lymphocytic leukemia or itstherapy, respectively; and the compound of the invention (or aparticular form thereof) may be used similarly, or preferably used byoral administration, for the treatment of those diseases and conditionseither alone or in combination with another therapeutic agentcustomarily indicated for the treatment of the particular condition. Fortherapeutic or prophylactic treatment of a vascular disease or relatedcondition in a mammal in which neutrophils are involved or implicated, acompound of the invention (or a particular form thereof) mayconveniently be administered by an oral or parenteral route, eitheralone or simultaneously or sequentially with other therapeuticallyactive agents customarily administered for the condition. The utility ofthe compound of the invention (or a particuler form thereof) in suchtreatment of vascular diseases and related conditions may bedemonstrated using the procedures described in International PatentApplication, Publication No. WO 92/22309.

The various aspects of the invention will now be illustrated by thefollowing non-limiting examples in which, unless stated otherwise:

(i) temperatures are given in degrees Celsius (°C.); operations werecarried out at room or ambient temperature, that is, at a temperature inthe range of 18°-25° C.;

(ii) organic solutions were dried over anhydrous magnesium sulfate;evaporation of solvent was carried out using a rotary evaporator underreduced pressure (600-4000 pascals; 4.5-30 mm Hg) with a bathtemperature of up to 60° C.;

(iii) chromatography means `flash chromatography` (method of Still)carried out on Merck Kieselgel (Art 9385 from E. Merck, Darmstadt,Germany), elution using both step and ramp gradients is denoted by theparenthetical term "gradient" followed by the initial and final solventratios; thin layer chomatography (TLC) was carried out on silica plates,for example 0.25 mm silica gel GHLF plates (Art 21521 from Analtech,Newark, Del., USA);

(iv) in general, the course of reactions was followed by TLC andreaction times are given for illustration only;

(v) melting points are uncorrected and (dec) indicates decomposition;the melting points given are those obtained for the materials preparedas described; polymorphism may result in isolation of materials withdifferent melting points in some preparations;

(vi) final products had satisfactory nuclear magnetic resonance (NMR)spectra; and, where examined, were substantially pure by HPLC;

(vii) yields are given for illustration only and are not necessarilythose which may be obtained by diligent process development;preparations were repeated if more material was required;

(viii) when given, NMR data is in the form of delta values for majordiagnostic protons, given in parts per million (ppm) relative totetramethylsilane (TMS) as an internal standard, determined at 250 MHzusing DMSO-d₆ as solvent; conventional abbreviations for signal shapeare used; for AB spectra the directly observed shifts are reported;

(ix) chemical symbols have their usual meanings; SI units and symbolsare used;

(x) reduced pressures are given as absolute pressures in pascals (Pa);elevated pressures are given as gauge pressures in bars;

(xi) solvent ratios are given in volume:volume (v/v) terms;

(xii) mass spectra (MS) were run with an electron energy of 70 electronvolts in the chemical ionizaton mode using a direct exposure probe;where indicated ionization was effected by electron impact (EI) or fastatom bombardment (FAB); generally, only peaks which indicate the parentmass are reported; and

(xiii) HPLC was used to establish the ratio of SSS:SSR diastereoisomersof formula I in isolated material, using a SUPELCO LC-18 reversed-phase,25 cm×4.6 mm column and water:acetonitrile (70:30) as eluant. The flowrate was 1.0 ml/min, the injection volume was 20 μl by valve and thedetection wavelength was 205 nm. The retention time for the SSSdiastereisomer was about 9.9 minutes and that for the SSRdiastereoisomer was about 11.7 minutes.

EXAMPLE 1

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.84 g) wasadded to a solution of (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(2,2,2-trifluoro-1-hydroxyethyl)propyl!pyrrolidine-2-carboxamide(0.41 g) dissolved in dimethylsulphoxide (DMSO; 5 ml) and toluene (5ml), followed by dropwise addition of dichloroacetic acid (0.32 ml). Theresulting solution was allowed to stir at 20° C. for 2 hours. Thesolution was then poured into ethyl acetate (200 ml) and washedsuccessively with 1M hydrochloric acid, water and brine. The organicsolution was dried (MgSO₄) and concentrated under vacuum. The residuewas purified by flash chromatography (gradient elution;methanol:methylene chloride, 3:97 to 5:95) to give (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(2,2,2-trifluoroacetyl)propyl!pyrrolidine-2-carboxamide (0.27g) as a white foam (as a mixture of ketone and hydrated forms); TLC,R_(f) =0.4 (methanol:dichloromethane, 2.5:97.5); ¹ H NMR (DMSO/D₂ O):4.44 (m, 1H), 4.00 (m, 2H), 3.72 (m, 1H), 3.51 (m, 4H), 202-1.75 (m,6H), 0.95-0.78 (m, 12H); Analysis for C₁₈ H₂₈ F₃ N₃ O₅ 0.3H₂ O:Calculated: C, 50.42; H, 6.72; N, 9.80; Found: C, 50.31; H, 6.28; N,9.56.

The starting material (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(2,2,2-trifluoro-1-hydroxyethyl)propyl!-pyrrolidine-2-carboxamidewas obtained as follows:

Methyl chloroformate (0.12 ml) was added to a solution of(2RS,3SR)-L-valyl-N-3-(4-methyl-1,1,1-trifluoro-2-hydroxypentyl)!-L-prolinamide (obtained asdescribed in U.S. Pat. No. 5,194,588) (0.5 g) and triethylamine (0.57ml) in dichloromethane (13.6 ml) at 0° C. The solution was allowed tostir for 0.5 hours, and then poured into ethyl acetate (100 ml). Theorganic solution was washed successively with saturated aqueous sodiumbicarbonate solution, water and brine. The solution was dried (MgSO₄)and concentrated under vacuum. The residue was purified by flashchromatography (gradient elution; methanol:methylene chloride, 5:95 to7:93) to give (S)-1- (S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(2,2,2-trifluoro-1-hydroxyethyl)propyl!pyrrolidine-2-carboxamide(0.51 g); TLC, R_(f) =0.2 (methanol:methylene chloride, 5:95); MS:m/z=426(M+1).

EXAMPLE 2

A solution of potassium permanganate (16.6 g) in water (100 ml) wasadded dropwise to a 0° C. solution of (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(S)-2-methyl-1-((R)-2,2,2-trifluoro-1-hydroxyethyl)propyl!pyrrolidine-2-carboxamide(15 g) in tert-butyl alcohol (175 ml), water (100 ml), and 0.6M sodiumhydroxide solution (175 ml). The solution was stirred for 2 hours andthen quenched by addition of methanol (70 ml), followed by stirring for1 hour. The mixture was filtered through diatomaceous earth and thefiltrate made acidic to pH 2 using 1M hydrochloric acid, and saturatedwith sodium chloride. The product was extracted into ether (5×100 ml)and the solvent removed under vacuum. The resulting oil waschromatographed (methanol: dichloromethane 5:95) and the solvent removedto give an oil. Hexane (40 ml) was added to a stirred solution of theoil in ethyl acetate (which had been presaturated with water) (40 ml)and stirring was continued for 24 hours over which time a crystallinesolid formed. Another portion of hexane (40 ml) was added and the solidwas collected and dried under vacuum (40° C.) to give (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(S)-2-methyl-1-(2,2,2-trifluoroacetyl)propyl!-pyrrolidine-2-carboxamide(9.45 g) as a white crystalline solid (as substantially or essentiallyForm A); ¹ H NMR (300 MHz, DMSO/D₂ O): 4.42 (m, 1H) 4.02 (d, 1H), 3.73(m, 1H), 3.59 (m, 1H), 3.54 (s, 3H), 2.23 (m, 1H), 2.00-1.76 (m, 6H),0.91 (m, 6H), 0.85 (d, 3H), 0.80 (d, 3H); Analysis for C₁₈ H₂₈ F₃ N₃O₅.H₂ O: Calculated; C, 48.97; H, 6.85; N, 9.51; Found: C, 49.02, H,6.80; N, 9.66; (XDS shown in FIG. 1).

The starting material (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(S)-2-methyl-1-((R)-2,2,2-trifluoro-1-hydroxyethyl)propyl!pyrrolidine-2-carboxamidewas obtained as follows:

(i) N- (Phenylmethoxy)carbonyl!-L-valyl-L-proline tert-butyl ester (905g) was dissolved in ethanol (4 liters) and 10% palladium on carbon (20g) was added. The reaction mixture was shaken under a hydrogenatmosphere (50 psi) for 12 hours and then the catalyst was removed byfiltration through through diatomaceous earth. The filtrate wasconcentrated under vacuum and the residue twice re-evaporated fromtoluene (1 liter) to give L-Valyl-L-Proline tert-butyl ester as an oil(628 g); TLC, R_(f) =0.2, acetone:hexane (20:80); MS: m/z=271(M+1).

(ii) A solution of sodium carbonate (110.5 g) in water (1.5 liters) andL-Valyl-L-Proline tert-butyl ester in tetrahydrofuran (THF; 1 liter)were combined and cooled to 0° C. The mixture was diluted with ether(400 ml) and methyl chloroformate (39.4 g) was added dropwise. Thereaction mixture was then allowed to warm ambient temperature over 2hours. The layers were separated and the organic phase was washed twicewith 1M hydrochloric acid, followed by saturated aqueous sodiumbicarbonate solution and brine. The aqueous phase was extracted withether. All organic phases were combined and dried (MgSO₄) and thesolvent removed to give N-(methoxycarbonyl)-L-valyl-L-proline tert-butylester (125.9 g); ¹ H NMR (300 MHz, DMSO/d4-trifluoroacetic acid): 4.23(dd, 1H), 4.06 (d, 1H), 3.78 (m, 1H), 3.57 (m, 1H), 3.55 (s, 3H), 2.16(m, 1H), 1.95 (m, 3H), 1.80 (m, 1H), 1.42 (s, 9H), 0.94 (m, 6H); MS:m/z=329 (M+1).

(iii) To a solution of N- methoxycarbonyl!-L-valyl-L-proline tert-butylester (813 g) in toluene (3 liters) was added Amberlyst-15 ion exchangeresin (190 g). The reaction was heated at 120° C. to distill off waterpresent in the resin via a water/toluene azeotrope. Approximately 400 mlof distillate was collected. Heating was then continued at reflux for1.5 hours. The reaction was cooled to 60° C. and the resin was removedby filtration. The filtrate was extracted with 1M NaOH (2.5 liters)followed by saturated aqueous sodium bicarbonate solution. The combinedbasic extracts were extracted with a mixture of THF/ethyl acetate (1:1,1 liter), and then cooled in an ice bath. The aqueous solution was madeacidic to pH 1.5 using cold 3M hydrocloric acid (1 liter) and extractedtwice with THF/ethyl acetate (1:1, 1.5 liters and 1 liter). The extractswere combined and washed with brine, dried (MgSO₄), and the solventremoved by evaporation. The resulting material was dissolved in ether (1liter) and allowed to crystallize at 0° C. over 48 hours. The resultingsolid was collected by filtration, washed with cold ether and driedunder vacuum to give N- methoxycarbonyl!-L-valyl-L-proline (373 g); ¹ HNMR (300 MHz, DMSO) 12.4 (s, 1H), 7.37 (d, 1H), 4.25 (dd, 1H), 4.00 (t,1H), 3.79 (m, 1H), 3.55 (m, 1H), 3.51 (s, 3H), 2.11 (m, 1H), 1.85 (m,4H), 0.91 (d, 3H), 0.87 (d, 3H); MS m/z=273 (M+1).

(iv) N-Methylmorpholine (8.5 ml) was added to a solution ofN-(methoxycarbonyl)-L-valyl-L-proline (12.5 g) in THF (150 ml) and thesolution was cooled to -15° C. in an ice/acetone bath. Isobutylchloroformate (6.6 ml) was added dropwise and the mixture was stirredfor 1 hour. A second portion of N-methylmorpholine (8.5 ml) was added,followed by (2R,3S)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanolhemioxalate salt (10 g). The reaction mixture was then allowed to stirfor 12 hours while allowing to warm to ambient temperature. The reactionmixture was diluted with ether (500 ml) and washed successively withsaturated aqueous sodium bicarbonate solution, 1M HCl and brine. Theaqueous layers were extracted with ether and all organic phases werecombined and dried (MgSO₄). The solution was filtered and the solventremoved by evaporation. The resulting material was filtered throughsilica gel using ether as the eluant. The ether fractions containing theproduct were combined and the solvent removed by evaporation. Theproduct was dried under vacuum to give (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(S)-2-methyl-1-(R)-(2,2,2-trifluoro-1-hydroxyethyl)propyl!-pyrrolidine-2-carboxamide(16.1 g); ¹ H NMR (300 MHz, DMSO): 7.61 (d, 1H), 7.28 (d, 1H), 6.44 (d,1H), 4.44 (m, 1H), 4.05 (m, 1H), 3.98 (m, 1H), 3.75 (m, 2H), 3.55 (m,1H), 3.50 (s, 3H), 1.83 (m, 6H), 0.90 (d, 3H), 0.86 (d, 3H); MS:m/z=426.

(2R,3S)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanol hemioxalate salt,used in step (iv), was obtained as follows:

(i) Triphosgene (23 g) was added in one portion to a well stirredmixture of (2RS,3SR)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanolhemioxalate salt (50 g) in toluene (250 ml) and 2M sodium hydroxidesolution (350 ml). The reaction began to exotherm and was placed in anice bath. After 0.5 hour the reaction was warmed to 25° C. and TLCindicated a substantial amount of unreacted amine present. The pH of thesolution was readjusted to about 12 using 50% sodium hydroxide solution.An additional portion of triphosgene (8 g) was added and the solutionwas stirred for 1 hour. The pH of the reaction mixture was lowered to pH7 using 1M hydrochloric acid and extracted twice with ether. Thecombined ether layers were washed with water, brine and dried (MgSO₄).The solvent was removed by evaporation to give an oil, whichcrystallized upon standing. The resulting solid was collected byfiltration and washed with ether:hexane (1:1) to give 27 g of(4RS),5SR)-4-isopropyl-5-trifluoromethyloxazolidin-2-one as a whitesolid, m.p. 71°-72° C.; ¹ H NMR (300 MHz, DMSO): 8.45 (s, 1H), 5.11 (m,1H), 3.61 (m, 1H), 1.72 (m, 1H), 0.86 (d, 6H).

(ii) n-Butyllithium (20 ml of a 10M solution in hexane) was added to asolution of (4RS),5SR)-4-isopropyl-5-trifluoromethyloxazolidin-2-one(35.8 g) in THF (600 ml) at -78° C., followed by stirring for 0.5 hours.(-)-Menthyl chloroformate (41 ml, freshly distilled) was added followedby continuation of stirring at -78° C. for 0.5 hours. The solution waswarmed to 25° C. and the reaction quenched by addition of saturatedaqueous sodium bicarbonate solution. The product was extracted intoether and washed with water and brine. The solution was dried (MgSO₄)and the solvent removed under vacuum. The resulting oil crystallizedupon standing to give a solid which was collected by filtration. Thesolid was washed with ether:hexane (1:1) and dried to give(4S,5R)-4-isopropyl-3-(1R,3R,4S)-3-p-menthyloxycarbonyl!-5-trifluoromethyloxazolidin-2-one(23.15 g); m.p. 138°-140° C.; ¹ H NMR (300 MHz, DMSO): 5.51 (dd, 1H),4.68 (m, 1H), 4.26 (m, 1H), 2.27 (m, 1H), 1.94 (d, 1H), 1.78 (m, 1H),1.62 (d, 2H), 1.42 (m, 2H), 1.01 (dd, 2H), 0.95-0.84 (m, 24H), 0.71 (d,3H); ¹⁹ FNMR (376.5 MHz,DMSO): -76.9910; 99% d.e. (A further crop of 4.3g (99% d.e.) was obtained from the mother liquor). Note: the (4R,5S)isomer has m.p. 80°-82° C. and 19FNMR (376.5 MHz, DMSO): -77.0019.

(iii) A solution of (4S,5R)-4-isopropyl-3-1(1R,3R,4S)-3-p-menthyloxycarbonyl!-5-trifluoromethyloxazolidin-2-one(27 g) in dioxane (70 ml) and 50% potassium hydroxide solution (80 ml)was heated at 100° C. for 2 days. The reaction was cooled, diluted withether (400 ml) and the organic layer separated. The pH of the aqueoussolution was adjusted to 9 (originally about 14) using 6M hydrochloricacid. The aqueous layer was extracted 3 times with ether (300 ml). Theorganic phases were combined, dried (MgSO₄), and added to a well stirredsolution of oxalic acid dihydrate (4.5 g) in acetonitrile (100 ml). Thesolid which precipitated was collected by filtration, washed with ether,and dried under vacuum (60° C.) to give 15.9 g of white solid. The solidwas triturated with ether (300 ml), collected by filtration and dried togive (2R,3S)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanol isolated as itshemioxalate salt (13.4.g, 88% yield) as a white solid, m.p. 184°-186°C.; ¹ H NMR (300 MHz, DMSO): 5.71 (bs, 3H), 4.08 (ddd, 1H), 2.88 (m,1H), 1.81 (m, 1H), 0.92 (m, 6H); Analysis for C₆ H₁₂ F₃ NO.0.5C₂ H₂ O₄ :C, 38.89; H, 6.06; N, 6.48; Found: C, 38.75; H, 5.95; N, 6.47.

(2RS,3SR)-3-Amino-4-methyl-1,1,1-trifluoro-2-pentanol used in step (i)was obtained as described in U.S. Pat. No. 4,910,190.

EXAMPLE 3

Using a similar oxidation procedure to that described in Example 2, butusing (S)-1- (S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(2,2,2-trifluoro-1-hydroxyethyl)propyl!-pyrrolidine-2-carboxamideand adding the potassium permanganate solution at 5°-10° C. and thenstirring at 10° C. for one hour prior to treatment with methanol, therewas obtained (after work-up by extration into tert-butyl methyl ether,followed by washing with brine and concentration in vacuo) (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(2,2,2-trifluoroacetyl)propyl!-pyrrolidine-2-carboxamide as agum (in 75% yield); SSS:SSR ratio of 53:47; hydrate:ketone 1:1; ¹ H NMRsimilar to that of the product of Example 1. Using a similar procedure,but adding the potassium permanganate solution at ambient temperatureinstead of 5°-10° C., the product was obtained in a ratio of SSS:SSR of47:53.!

The starting material (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(2,2,2-trifluoro-1-hydroxyethyl)-propyl!pyrrolidine-2-carboxamidewas obtained as an oil (in 55% yield) using an analogous procedure tothat described in Example 2, part (iv), but using3-amino-4-methyl-1,1,1-trifluoro-2-pentanol (as a mixture ofdiastereoisomers), itself obtained as described in U.S. Pat. No.4,910,190 or as follows:

(i) A solution of urea (72 g) in DMF (810 ml) was added to sodiumnitrite (90 g), stirred for 10 minutes and then cooled to 15° C.Isobutyl iodide (97.2 ml) was added over 30 minutes and the reactionmixture allowed to stir at ambient temperature for 20 hours. The mixturewas re-cooled to 15° C. and water (810 ml) was added slowly. The mixturewas was stirred for 5 minutes at ambient temperature and then extractedtwice with methyl tert-butyl ether. The combined organic extracts werewashed twice with 20% aqueous sodium thiosulphate solution andconcentrated under vacuum to give 2-methyl-1-nitropropane (39 g), whichwas used without further purification.

(ii) 3A Molecular sieves (27.04 g) were heated at 120° C. under vacuumfor 20 hours and added to a solution of 2-methyl-1-nitropropane (13.0 g)in methyl tert-butyl ether (420 ml). The mixture was stirred for 5minutes, potassium carbonate (64.5 g) added and the mixture stirred afurther 30 minutes. The mixture was cooled to 15° C. and fluoral hydrate(22.0 g) was added over 30 minutes. The reaction mixture was stirred atambient temperature for 16 hours, then cooled to 15° C. and water (270ml) added. After stirring for 5 minutes at ambient temperature, theorganic phase was separated and washed with 10% aqueous potassiumcarbonate, 2M hydrochloric acid solution and water. Solvent was thenremoved by evaporation under reduced pressure at a temperature below 40°C. and the oil azeotroped dry with isopropyl alcohol at a temperaturebelow 50° C. to give 4-methyl-3-nitro-1,1,1-trifluoro-2-pentanol (21.3g) as an oil, which was used without further purification.

(iii) A solution of 4-methyl-3-nitro-1,1,1-trifluoro-2-pentanol (17.1 g)in isopropanol (115 ml) and acetic acid (0.43 ml) was hydrogenated over10% palladium on carbon (2.4 g) at 3.5 bar pressure until uptake ofhydrogen was complete. The catalyst was removed by filtration throughdiatomaceous earth and the filter cake washed with isopropanol. Thefiltrate was evaporated under vacuum until no further isopropanoldistilled and the residue dissolved in acetonitrile (40 ml). A solutionof oxalic acid (3.94 g) in acetonitrile (80 ml) was added with stirringand the mixture cooled to 5° C. The product which crystallised wascollected by filtration, washed with cold acetonitrile and dried at 50°C. to give 3-amino-4-methyl-1,1,1-trifluoro-2-pentanol as its oxalatesalt (9.08 g).

EXAMPLE 4

Hexane (13 ml) was added to a solution of (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-2-methyl-1-(2,2,2-trifluoroacetyl)propyl!pyrrolidine-2-carboxamide (0.85g; SSS:SSR 53:47; hydrate:ketone 1:1) in tert-butyl methyl ether (8.5ml) until cloudiness persisted. The solution was then warmed to give aclear solution, seeded with substantially pure crystalline SSSdiastereoisomer and allowed to stand. A white solid crystallised whichwas collected by filtration to give (S)-1-(S)-2-(methoxycarbonylamino)-3-methylbutyryl!-N-(S)-2-methyl-1-(2,2,2-trifluoroacetyl)propyl!-pyrrolidine-2-carboxamideas a crystalline solid in 30% yield, SSS:SSR 95:5; hydrate:ketone 80:20;NMR similar to that of the product of Example 2.

EXAMPLE 5

Using a similar procedure to that described in Example 4, but startingwith a diastereomeric mixture of SSS:SSR 53:47 (1.73 g) andhydrate:ketone 95:5, but adding 36% w/w hydrochloric acid (0.06 ml) andwater (0.04 ml) to the crystallisation solvent prior to the addition ofthe hexane, crystalline SSS diastereisomer was obtained in 22% yieldwith SSS:SSR 98.5:1.5 and substantially or essentially in a hydrateform.

EXAMPLE 6

Using a similar procedure to that described in Example 5, but excludingthe hydrochloric acid, a crystalline diastereomeric mixture was obtainedwith SSS:SSR 65:35 and substantially or essentially in a hydrate form.

EXAMPLE 7

Using a similar procedure to that described in Example 5, but startingwith a diastereomeric mixture of SSS:SSR 47:53 and hydrate:ketone 60:40,crystalline SSS diastereoisomer was obtained in 18% yield with SSS:SSR98.5:1.5 and substantially or essentially in a hydrate form.

EXAMPLE 8

The product of Example 2 (5 g) was dissolved in 1,2-dimethoxyethane(DME; 6 ml) with slight warming. Water (5 ml) was carefully added to thesolution to give a clear solution. The solution was allowed to cool toambient temperature, seeded with substantially pure crystalline SSSdiastereoisomer and allowed to stand for 16 hours. The crystalline masswhich had formed in the bottom of the vessel was carefully broken up andcollected by vacuum filtration. The crystalline product was washed witha mixture of DME and water and allowed to dry in a current of air for 16hours to give crystalline SSS diastereoisomer (containing less then 2%SSR diastereoisomer) as substantially or essentially Form B, with awater content of 7.3% v/w; (XDS spectrum shown in FIG. 3). Using asimilar procedure but using recrystallised Form A as starting material,Form B was obtained having a water content of 7.7% w/w.!

EXAMPLE 9

The product of Example 8 (4.78 g) was dissolved in ethyl acetate (14.7ml) with warming to 60° C. under an inert atmosphere. Hexane (22 ml) wasadded slowly and the solution was allowed to cool to 22° C. Thecrystalline product was collected by filtration and washed with hexane(10 ml), then allowed to dry in a current of air to give crystalline SSSdiastereoisomer (containing less then 2% SSR diastereoisomer) assubstantially or essentially Form A, with a water content of 4.1% w/w.

EXAMPLE 10

The product of Example 2 (1 g) was dissolved in cyclohexane (20 ml) andthe solution was distilled at atmospheric pressure at 80° C. to reducethe volume to 7 ml. The clear solution was then allowed to cool to 24°C. The suspended solid was collected by suction filtration carried outunder a current of dry nitrogen and dried in a desiccator under vaccumin the presence of phosphorus pentoxide. There was thus obtainedcrystalline SSS diastereoisomer (containing less then 2% SSRdiastereoisomer) in substantially or essentially the "ketone" form; (XDSspectrum shown in FIG. 4).

    ______________________________________                                                                    Chemical                                                                      Formulae                                          ______________________________________                                         ##STR1##                   I                                                  ##STR2##                   Ia                                                 ##STR3##                   Ib                                                 ##STR4##                   Ic                                                 ##STR5##                                                                      ##STR6##                   II                                                 ##STR7##                   IIa                                               ______________________________________                                         ##STR8##                                                                       Suitable conditions include: (a) DMF, 1-hydroxybenztriazole, Et.sub.3 N,       dicyclohexylcarbodiimide, 0° C. to ambient temperature             

(b) R=^(t) Bu: trifluoroacetic acid, CH₂ Cl₂, 0° C. to ambienttemperature R=Me: methanol/aqueous NaOH, ambient temperature

(c) ^(i) BuO.CO.Cl, N-methylmorpholine, THF, -35° C. to 0° C., followedby the aminoalcohol

(d) H₂, 10% Pd-C, EtOH

(e) CH₃ O.CO.Cl, Et₃ N, CH₂ Cl₂, 0° C. ##STR9## Suitable conditions forScheme 2 include: Steps (1), (8), (9): as for Step (d) of Scheme 1;Steps (2), (7), (11): HeOCOCl, Et₃ N or N-methylmorpholine, CH₂ Cl₂ orTHF, 0° C. to 30° C.; Steps (3), (6): trifluoroacetic acid, CH₂ Cl₂, 0°C. to ambient temp; Steps (4), (5): as for Step (a) of Scheme 1; Step(10): Me₃ SiCl, THF, N-methylmorpholine, 0°-30° C.; Step (12): acidicaqueous hydrolysis; Steps (13), (14): as for step (c) of Scheme 1##STR10##

What is claimed is:
 1. A process for the manufacture of a compound, offormula Ior a solvated form thereof, as a diastereomeric mixturecomprising 50% or more of the diastereoisomer of formula Ia ##STR11## ora solvated form thereof, which comprises oxidation of a compound offormula II ##STR12## with a suitable oxidising agent.
 2. A process asclaimed in claim 1 wherein a compound of formula IIa ##STR13## is usedas starting material.
 3. A process as claimed in claim 2, followed byforming crystals from a solution of the product in a solvent selectedfrom butyl acetate, a mixture of butyl acetate and hexane, a mixture ofacetone and water, a mixture of acetone and hexane, a mixture of acetoneand petroleum ether b.p. 100°-120° C., a mixture of 1,2-dimethoxyethaneand hexane, a mixture of 1,2-dimethoxyethane, water and hexane, amixture of ethyl acetate and hexane, a mixture of ethyl acetate, hexaneand water, water, a mixture of dibutyl ether and hexane, a mixture ofdichloromethane and hexane, a mixture of methanol and toluene, a mixtureof tert-butyl methyl ether and hexane, a mixture of isopropanol andhexane and a mixture of tetrahydrofuran and hexane.
 4. A process for themanufacture of the intermediate(2R,3S)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanol, or a salt thereof,which comprises(i) reaction of(4RS,5SR)-4-isopropyl-5-trifluoromethyloxazolidin-2-one, or an alkalimetal salt thereof, with (-)-menthyl chloroformate to give (4RS,5SR)-4-isopropyl-3-(1R,3R,4S)-3-p-menthyloxycarbonyl!-5-trifluoromethyloxazolidin-2-one;(ii) separation of the (4S,5R)-4-isopropyl-3-(1R,3R,4S)-3-p-menthyloxycarbonyl!-5-trifluoromethyloxazolidin-2-oneisomer; and (iii) hydrolysing the (4S,5R)-4-isopropyl-3-(1R,3R,4S)-3-p-menthyloxycarbonyl!-5-trifluoromethyloxazolidin-2-oneisomer under basic conditions to give(2R,3S)-3-amino-4-methyl-1,1,1-trifluoro-2-pentanol.
 5. A process forpreparing a compound of formula Ib ##STR14## which is crystalline and isin hydrated form and has an X-ray powder diffraction pattern (a)including specific peaks at about 2-theta=10.8, 11.4, 15.4, 21.6 and21.9 degrees of (b) including specific peaks at about 2-theta=7.2, 7.4,9.0, 9.2, 10.8, 11.3, 14.5, 15.9, 17.8, 19.7 and 22.5 degrees whichcomprises forming crystals from a solution of substantially oressentially pure diastereoisomer of formula Ia in a hydrated form in asolvent selected from butyl acetate, a mixture of butyl acetate andhexane, a mixture of acetone and water, a mixture of acetone and hexane,a mixture of acetone and petroleum ether b.p. 100°-120° C., a mixture of1,2-dimethoxyethane and hexane, a mixture of 1,2-dimethoxyethane, waterand hexane, a mixture of ethyl acetate and hexane, a mixture of ethylacetate, hexane and water and water.